[Identification of key enzyme genes involved in biosynthesis pathways of lignan and lignin in Eucommia ulmoides based on transcriptome assembly].

Lignan is the main medicinal component of Eucommia ulmoides, and lignin is involved in the defense of plants against diseases and insect pests.They are synthesized from coniferyl alcohol with the help of dirigent(DIR) and peroxidase(POD), respectively.In this study, transcriptome assembly of stems and leaves of E.ulmoides was performed, yielding 112 578 unigenes.Among them, 70 459 were annotated in seven databases.A total of 59 unigenes encodes 11 key enzymes in the biosynthesis pathways of lignin and lignin, of which 11 encode POD and 8 encode DIR.A total of 13 unigenes encoding transcription factors are involved in phenylpropanoid metabolism. Compared with leaves of E.ulmoides, 7 575 unigenes were more highly expressed in stems, of which 462 were involved in phenylpropanoid biosynthesis.Our results extend the public transcriptome dataset of E.ulmoides, which provide valuable information for the analysis of biosynthesis pathways of lignan and lignin in E.ulmoides and lay a foundation for further study on the functions and regulation mechanism of key enzymes in lignan and lignin biosynthesis pathways.

Comparative transcriptomic analysis reveals the regulatory mechanisms of catechins synthesis in different cultivars of Camellia sinensis.

Camellia sinensis (L.) O. Kuntze is used to produce tea, a beverage consumed worldwide. Catechins are major medically active components of C. sinensis and can be used clinically to treat hyperglycaemia, hypertension, and cancer. In this study, we aimed to identify the genes involved in catechins biosynthesis. To this end, we analysed transcriptome data from two different cultivars of C. sinensis using DNBSEQ technology. In total,47,717 unigenes were obtained from two cultivars of C. sinensis, of which 9429 were predicted as new unigenes. In our analyses of the Kyoto Encyclopedia of Genes and Genomes database, 212 unigenes encoding 13 key enzymes involved in catechins biosynthesis were identified; the structures of leucoanthocyanidin reductase and anthocyanidin reductase were spatially modelled. Some of these key enzymes were verified by real-time quantitative polymerase chain reaction, and multiple genes encoding plant resistance proteins or transcription factors were identified and analysed. Furthermore, two microRNAs involved in the regulation of catechins biosynthesis were explored. Differentially expressed genes involved in the flavonoid biosynthesis pathway were identified from pairwise comparisons of genes from different cultivars of tea plants. Overall, our findings expanded the number of publicly available transcript datasets for this valuable plant species and identified candidate genes related to the biosynthesis of C. sinensis catechins, thereby establishing a foundation for further in-depth studies of catechins biosynthesis in varieties or cultivars of C. sinensis.

Probing the transcriptome of Boehmeria nivea reveals candidate genes associated with the biosynthesis of chlorogenic acid.

Boehmeria nivea (L.) Gaudich is used in traditional Chinese medicine. Chlorogenic acids are major medically active components of Boehmeria nivea, which can be used clinically to treat hyperglycemia, pneumonia, and cancer. To identify the genes involved in chlorogenic acid biosynthesis, we analyzed transcriptome data from leaf, root, and stem tissues of Boehmeria nivea using the Illumina Hi-Seq 4000 platform. A total of 146,790 unigenes were obtained from Boehmeria nivea, of which 106,786 were annotated in public databases. In analyses of the KEGG (Kyoto Encyclopedia of Genes and Genome) database, 484 unigenes that encode the five key enzymes involved in chlorogenic acid biosynthesis were identified, and shikimate O-hydroxycinnamoyl transferase was spatially simulated. Some of these key enzyme unigenes expression levels were verified by RT-qPCR (real-time quantitative Polymerase Chain Reaction). Furthermore, multiple genes encoding plant resistance proteins or transcription factors were identified and analyzed. Differentially expressed genes were identified by performing pairwise comparison of genes between tissues. This study increases the number of public transcript datasets of this species and identifies candidate genes related to the biosynthesis of chlorogenic acid, laying a foundation for the further exploration of this pathway in Boehmeria nivea.

Transcriptome analyses reveal the expression profile of genes related to lignan biosynthesis in Anthriscus sylvestris L. Hoffm. Gen.

Anthriscus sylvestris L. Hoffm. Gen (A. sylvestris) is a perennial herb widely used for antitussive and diuretic purposes in traditional Korean and Chinese medicine. Lignans are critical secondary metabolites with widely pharmacological activities in A. sylvestris. Using transcriptome data of A. sylvestris, we identified genes related to lignan biosynthesis. In all, 123,852 unigenes were obtained from the flowers, leaves, roots, and stems of A. sylvestris with the Illumina HiSeq 4000 platform. The average length of unigenes was 1,123 bp and 91,217 (73.65%) of them were annotated in public databases. Differentially expressed genes and root-specific genes were analyzed between roots and the other three tissue types by comparing gene expression profiles. Specifically, the key enzyme genes involved in lignan biosynthesis were identified and analyzed. The expression levels of some of these genes were highest in the roots, consistent with the accumulation of deoxypodophyllotoxin. These expression levels were experimentally verified via quantitative real-time polymerase chain reaction (qRT-PCR). This research provides valuable information on the transcriptome data of A. sylvestris and the identification of candidate genes associated with the biosynthesis of lignans, laying the foundation for further research on genomics in A. sylvestris and related species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01156-w.

Exploring the biosynthetic pathway of lignin in Acorus tatarinowii Schott using de novo leaf and rhizome transcriptome analysis.

Acorus tatarinowii Schott is a well-known Chinese traditional herb. Lignin is the major biologically active ingredient and exerts a broad range of pharmacological effects: it is an antitumor, antioxidant and bacteriostatic agent, and protects the cardiovascular system. In the present study, the transcriptomes of the leaf and rhizome tissues of A. tatarinowii Schott were obtained using the BGISEQ-500 platform. A total of 141777 unigenes were successfully assembled, of which 76714 were annotated in public databases. Further analysis of the lignin biosynthesis pathway revealed a total of 107 unigenes encoding 8 key enzymes, which were involved in this pathway. Furthermore, the expression of the key genes involved in lignin synthesis in different tissues was identified by quantitative real-time PCR. Analysis of the differentially expressed genes (DEGs) showed that most of the up-regulated unigenes were enriched in rhizome tissues. In addition, 2426 unigenes were annotated to the transcriptome factor (TF) family. Moreover, 16 TFs regulating the same key enzyme (peroxidase) were involved in the lignin synthesis pathway. The alignment of peroxidase amino acid sequences and the analysis of the structural characteristics revealed that the key peroxidase enzyme had well-conserved sequences, spatial structures, and active sites. The present study is the first to provide comprehensive genetic information on A. tatarinowii Schott at the transcriptional level, and will facilitate our understanding of the lignin biosynthesis pathway.

De novo transcriptome assembly of Angelica dahurica and characterization of coumarin biosynthesis pathway genes.

Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav (A. dahurica) is a famous Chinese herb known for the production of coumarins, important secondary metabolites with wide-ranging pharmacological activities. In particular, the methoxylated coumarins like those produced by A. dahurica are known for their anti-inflammatory, anti-cancer, and anti-oxidant pharmacological effects. However, the molecular mechanism of coumarin biosynthesis in A. dahurica has not been studied. Such investigation could help scientists harness the biosynthesis potential of methoxylated coumarins. Here we present, three transcriptomes corresponding to leaf, root, and stem tissues of A. dahurica. A total of 114,310 unigenes with an average length of 1118 bp were de novo assembled, and 81,404 (71.21%) of those unigenes were annotated. Then, 181 unigenes encoding the seven key enzymes involved were identified, for which COMT (Caffeic acid 3-O-methyltransferase) was spatially used in a phylogenetic analysis, and some of these key enzyme genes were verified by qRT-PCR. Differentially expressed genes and root-specific-expressed genes were identified, by comparing genes' profile activity between roots and other tissues. Furthermore, multiple genes encoding key enzymes or transcription factors related to coumarin biosynthesis were identified and analyzed. This study is the first to report comprehensive gene information of A. dahurica at the transcriptional level, and to distinguish candidate genes related to its biosynthesis of coumarin, thus laying a foundation for this pathway's further exploration in A. dahurica.

De novo transcriptome analysis of Cnidium monnieri (L.) Cuss and detection of genes related to coumarin biosynthesis.

Cnidium monnieri (L.) Cuss (C. monnieri) is one of the most widely used traditional herbal medicines, exhibiting a wide range of pharmacological functions for treating asynodia, trichomonas vaginitis, and osphyalgia. Its important medicinal value comes from its abundance of coumarins. To identify genes involved in coumarin biosynthesis and accumulation, we analyzed transcriptome data from flower, leaf, root and stem tissues of C. monnieri. A total of 173,938 unigenes with a mean length of 1,272 bp, GC content of 38.79%, and N50 length of 2,121 bp were assembled using the Trinity program. Of these, 119,177 unigenes were annotated in public databases. We identified differentially expressed genes (DEGs) based on expression profile analysis. These DEGs exhibited higher expression levels in flower tissue than in leaf, stem or root tissues. We identified and analyzed numerous genes encoding enzymes involved in coumarin biosynthesis, and verified genes encoding key enzymes using quantitative real-time PCR. Our transcriptome data will make great contributions to research on C. monnieri and provide clues for identifying candidate genes involved in coumarin metabolic pathways.

[Identification of key enzyme genes involved in biosynthesis of steroidal saponins and analysis of biosynthesis pathway in Polygonatum cyrtonema].

Steroidal saponins, which are the characteristic and main active constituents of Polygonatum, exhibit a broad range of pharmacological functions, such as regulating blood sugar, preventing cardiovascular and cerebrovascular diseases and anti-tumor. In this study, we performed RNA sequencing(RNA-Seq) analysis for the flowers, leaves, roots, and rhizomes of Polygonatum cyrtonema using the BGISEQ-500 platform to understand the biosynthesis pathway of steroidal saponins and study their key enzyme genes. The assembly of transcripts for four tissues generated 129 989 unigenes, of which 88 958 were mapped to several public databases for functional annotation, 22 813 unigenes were assigned to 53 subcategories and 64 877 unigenes were annotated to 136 pathways in KEGG database. Furthermore, 502 unigenes involved in the biosynthesis pathway of steroidal saponins were identified, of which 97 unigenes encoding 12 key enzymes. Cycloartenol synthase, the first key enzyme in the pathway of phytosterol biosynthesis, showed conserved catalytic domain and substrate binding domain based on sequence analysis and homology modeling. Differentially expressed genes(DEGs) were identified in rhizomes as compared to other tissues(flowers, leaves or roots).The 2 437 unigenes annotated by KEGG showed rhizome-specific expression, of which 35 unigenes involved in the biosynthesis of steroidal saponins. Our results greatly extend the public transcriptome dataset of Polygonatum and provide valuable information for the identification of candidate genes involved in the biosynthesis of steroidal saponins and other important secondary metabolites.

Transcriptome analysis of Clinopodium gracile (Benth.) Matsum and identification of genes related to Triterpenoid Saponin biosynthesis.

BACKGROUND: Clinopodium gracile (Benth.) Matsum (C. gracile) is an annual herb with pharmacological properties effective in the treatment of various diseases, including hepatic carcinoma. Triterpenoid saponins are crucial bioactive compounds in C. gracile. However, the molecular understanding of the triterpenoid saponin biosynthesis pathway remains unclear. RESULTS: In this study, we performed RNA sequencing (RNA-Seq) analysis of the flowers, leaves, roots, and stems of C. gracile plants using the BGISEQ-500 platform. The assembly of transcripts from all four types of tissues generated 128,856 unigenes, of which 99,020 were mapped to several public databases for functional annotation. Differentially expressed genes (DEGs) were identified via the comparison of gene expression levels between leaves and other tissues (flowers, roots, and stems). Multiple genes encoding pivotal enzymes, such as squalene synthase (SS), or transcription factors (TFs) related to triterpenoid saponin biosynthesis were identified and further analyzed. The expression levels of unigenes encoding important enzymes were verified by quantitative real-time PCR (qRT-PCR). Different chemical constituents of triterpenoid saponins were identified by Ultra-Performance Liquid Chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). CONCLUSIONS: Our results greatly extend the public transcriptome dataset of C. gracile and provide valuable information for the identification of candidate genes involved in the biosynthesis of triterpenoid saponins and other important secondary metabolites.

[Transcriptome analysis of venom gland and identification of functional genes for snake venom protein in Agkistrodon acutus].

Agkistrodon acutus is a traditional Chinese herb medicine which has immunological regulation,anti-tumor,anti-inflammatory and analgesic effects,which is mainly used for the treatment of rheumatoid arthritis,ankylosing spondylitis,sjogren's syndrome and tumors. In order to excavate more important functional genes from A. acutus,the transcriptome of the venom gland was sequenced by the Illumina Hi Seq 4000,and 32 862 unigenes were assembled. Among them,26 589 unigenes were mapped to least one public database. 2 695 unigenes were annotated and assigned to 62 TF families,and 5 920 SSR loci were identified. The majority of mapped unigenes was from Protobothrops mucrosquamatus in the NR database,which revealed their closest homology. Three secretory phospholipase A_2 with different amino acid sequences showed similar spatial structures and all had well-conserved active sites. The 3 D structural models of C-type lectin showed conserved glycosylation binding sites( Asn45). This study will lay the foundation for the further study of the function of snake venom protein,and promoting the development and utilization of genome resources from A. acutus.